Project Summary/ Abstract We propose a comprehensive investigation into the genetic basis of the development of vancomycin intermediate Staphylococcus aureus (VISA) and heterogeneous resistant S. aureus (hVISA) (collectively hVISA/VISA)). Identifying the regulatory and metabolic pathways that are altered in hVISA/VISA strains will be an important step toward more effective management of vancomycin monotherapy of patients infected with methicillin-resistant S. aureus (MRSA). The need is pressing. A 2007 report estimated that the number of MRSA infections in hospitals doubled nationwide, from approximately 127,000 in 1999 to 278,000 in 2005, and deaths increased from 11,000 to more than 17,000. In comparison, complications from the AIDS virus killed about 12,500 Americans in 2005. The rate of invasive MRSA rose to 31.8 cases per 100,000, costing the healthcare system billions of dollars. Vancomycin has been the most reliable therapeutic agent against MRSA for the past three decades. However, increasing use of vancomycin for Gram-positive organisms, including MRSA, has likely contributed to the growing burden of less susceptible strains and many healthcare facilities have reported an upward trend of vancomycin MICs for MRSA isolates over the past 5 years. The problem in pinning down hVISA/VISA is that the phenotype can be caused by multiple mutations in a complex network of interacting genes. Our first aim is to use the power of next generation sequencing technologies to characterize development of hVISA/VISA in the laboratory strain Newman, community-associated USA300 and a hospital infection strain. All strains will be tested for cross-resistance to an extended panel of drugs, including daptomycin and linezolid. More than 250 laboratory mutants of vancomycin susceptible (VSSA) S. aureus strains will be screened by resequencing. The causality of the principal variants discovered will be confirmed by genetic reconstruction of the mutants. Our second aim is then to determine the genetic basis of resistance in a diverse collection of 250 clinical strains from CDC and other epidemiologic studies that have already been accurately defined as VISA or hVISA. The final aim is to disentangle the complexity of genetic variation into discrete classes of transcriptional regulation using the RNA-Seq next generation sequencing application. This work will lay the basis of discovering novel targets and choke points across the diversity of VISA strains and open the door to the design of genetic-based diagnostics.